LOCUS       sequence-472878-        7190 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV construct for HITI insert with Ckm  gRNA and hF9 transgene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7190)
  AUTHORS   Padmaswari MH, Bulliard G, Agrawal S, Jia MS, Khadgi S, Murach KA, 
            Nelson CE
  TITLE     Precision and efficacy of RNA-guided DNA integration in 
            high-expressing muscle loci.
  JOURNAL   Mol Ther Nucleic Acids. 2024 Sep 2;35(4):102320. doi: 
            10.1016/j.omtn.2024.102320. eCollection 2024 Dec 10.
  PUBMED    39398225
REFERENCE   2  (bases 1 to 7190)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Ther 
            Nucleic Acids."; date: "2024-09-2"; volume: "35(4):102320. doi"; 
            pages: " 10.1016/j.omtn.2024.102320. eCollection 2024 Dec 10"
COMMENT     Sequence Label: pAAV_Ckm_F9
FEATURES             Location/Qualifiers
     source          1..7190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     79..100
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(254..273)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     373..395
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(433..451)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        519..623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(624..692,693..1484)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(842..861)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1655..2243
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2144..2163
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2305..2434
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        2462..2710
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2462..2482
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2633..2652
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2732..2807
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     polyA_signal    5770..5904
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5794..5813)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5848..5867
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     regulatory      6020..6029
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(6026..6028,6029..6031,6032..6745)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(6026..6028,6029..6031,6032..6742)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6071..6092)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6332..6351)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6679..6700
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   6757..6897
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6757..6886
                     /label=AAV2 ITR
     rep_origin      join(6972..7190,1..237)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7059..7078)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
       61 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
      121 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
      181 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
      241 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
      301 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
      361 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
      421 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
      481 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
      541 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
      601 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
      661 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
      721 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
      781 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
      841 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
      901 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
      961 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     1021 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     1081 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     1141 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     1201 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     1261 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     1321 ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc
     1381 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     1441 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     1501 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     1561 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     1621 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     1681 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     1741 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     1801 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     1861 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     1921 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     1981 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     2041 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     2101 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     2161 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     2221 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     2281 tttgctggcc ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg
     2341 ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga
     2401 gagggagtgg ccaactccat cactaggggt tcctgcggcc tctagactcg aattgggtac
     2461 cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     2521 gataattgga attaattcga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     2581 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     2641 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg
     2701 acgaaacacc gagtccccag caggatcaca tgttttagag ctagaaatag caagttaaaa
     2761 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttccgcgg
     2821 tggagagctg gagctcccct atgtgatcct gctggggact atgtccccta ggttcctggg
     2881 gagggaggac gtcggggcca gccaggctta ccccccctgc cgctgagact tctgcgctgg
     2941 tgcaccgcgc ctcttcgcgg tctccctgtc cttgcagaga accggtcagg ccaccatgca
     3001 gcgcgtgaac atgatcatgg cagaatcacc aggcctcatc accatctgcc ttttaggata
     3061 tctactcagt gctgaatgta cagtttttct tgatcatgaa aacgccaaca aaattctgaa
     3121 tcggccaaag aggtataatt caggtaaatt ggaagagttt gttcaaggga accttgagag
     3181 agaatgtatg gaagaaaagt gtagttttga agaagcacga gaagtttttg aaaacactga
     3241 aagaacaact gaattttgga agcagtatgt tgatggagat cagtgtgagt ccaatccatg
     3301 tttaaatggc ggcagttgca aggatgacat taattcctat gaatgttggt gtccctttgg
     3361 atttgaagga aagaactgtg aattagatgt aacatgtaac attaagaatg gcagatgcga
     3421 gcagttttgt aaaaatagtg ctgataacaa ggtggtttgc tcctgtactg agggatatcg
     3481 acttgcagaa aaccagaagt cctgtgaacc agcagtgcca tttccatgtg gaagagtttc
     3541 tgtttcacaa acttctaagc tcacccgtgc tgagactgtt tttcctgatg tggactatgt
     3601 aaattctact gaagctgaaa ccattttgga taacatcact caaagcaccc agtcatttaa
     3661 tgacttcact cgggttgttg gtggagaaga tgccaaacca ggtcaattcc cttggcaggt
     3721 tgttttgaat ggtaaagttg atgcattctg tggaggctct atcgttaatg aaaaatggat
     3781 tgtaactgct gcccactgtg ttgaaactgg tgttaaaatt acagttgtcg caggtgaaca
     3841 taatattgag gagacagaac atacagagca aaagcgaaat gtgattcgaa ttattcctca
     3901 ccacaactac aatgcagcta ttaataagta caaccatgac attgcccttc tggaactgga
     3961 cgaaccctta gtgctaaaca gctacgttac acctatttgc attgctgaca aggaatacac
     4021 gaacatcttc ctcaaatttg gatctggcta tgtaagtggc tggggaagag tcttccacaa
     4081 agggagatca gctttagttc ttcagtacct tagagttcca cttgttgacc gagccacatg
     4141 tcttcgatct acaaagttca ccatctataa caacatgttc tgtgctggct tccatgaagg
     4201 aggtagagat tcatgtcaag gagatagtgg gggaccccat gttactgaag tggaagggac
     4261 cagtttctta actggaatta ttagctgggg tgaagagtgt gcaatgaaag gcaaatatgg
     4321 aatatatacc aaggtatccc ggtatgtcaa ctggattaag gaaaaaacaa agctcactta
     4381 atgaaagatg gatttccaag gttaattcat tggaattgaa aattaacagg gcctctcact
     4441 aactaatcac tttcccatct tttgttagat ttgaatatat acattctatg atcattgctt
     4501 tttctcttta caggggagaa tttcatattt tacctgagca aattgattag aaaatggaac
     4561 cactagagga atataatgtg ttaggaaatt acagtcattt ctaagggccc agcccttgac
     4621 aaaattgtga agttaaattc tccactctgt ccatcagata ctatggttct ccactatggc
     4681 aactaactca ctcaattttc cctccttagc agcattccat cttcccgatc ttctttgctt
     4741 ctccaaccaa aacatcaatg tttattagtt ctgtatacag tacaggatct ttggtctact
     4801 ctatcacaag gccagtatca cactcatgaa gaaagaacac aggagtagct gagaggctaa
     4861 aactcatcaa aaacactact ccttttcctc taccctattc ctcaatcttt taccttttcc
     4921 aaatcccaat ccccaaatca gtttttctct ttcttactcc ctctctccct tttaccctcc
     4981 atggtcgtta aaggagagat ggggagcatc attctgttat acttctgtac acagttatac
     5041 atgtctatca aacccagact tgcttccgta gtggagactt gcttttcaga acatagggat
     5101 gaagtaaggt gcctgaaaag tttgggggaa aagtttcttt cagagagtta agttatttta
     5161 tatatataat atatatataa aatatataat atacaatata aatatatagt gtgtgtgtat
     5221 gcgtgtgtgt agacacacac gcatacacac atataatgga agcaataagc cattctaaga
     5281 gcttgtatgg ttatggaggt ctgactaggc atgatttcac gaaggcaaga ttggcatatc
     5341 attgtaacta aaaaagctga cattgaccca gacatattgt actctttcta aaaataataa
     5401 taataatgct aacagaaaga agagaaccgt tcgtttgcaa tctacagcta gtagagactt
     5461 tgaggaagaa ttcaacagtg tgtcttcagc agtgttcaga gccaagcaag aagttgaagt
     5521 tgcctagacc agaggacata agtatcatgt ctcctttaac tagcataccc cgaagtggag
     5581 aagggtgcag caggctcaaa ggcataagtc attccaatca gccaactaag ttgtcctttt
     5641 ctggtttcgt gttcaccatg gaacattttg attatagtta atccttctat cttgaatctt
     5701 ctagagagtt gctgaccaac tgacgtatgt ttccctttgt gaattaataa actggtgttc
     5761 tggttcatgg atccagacat gataagatac attgatgagt ttggacaaac cacaactaga
     5821 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     5881 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     5941 cagggggagg tgtgggaggt tttttagtcg acctatgtga tcctgctggg gactggtacc
     6001 caattcgcgc agaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
     6061 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     6121 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     6181 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     6241 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     6301 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     6361 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     6421 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     6481 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     6541 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     6601 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     6661 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     6721 cggcatggac gagctgtaca agtaaagcgg ccgcgcagga acccctagtg atggagttgg
     6781 ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     6841 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc
     6901 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     6961 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     7021 cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc
     7081 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     7141 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
//
